Germline and somatic mutations affecting various cell proliferation pathways are known to affect the development of cancer in patients. For example, the acquisition of somatic mutations that confer growth advantage on the cells possessing such mutations is considered an important factor in the emergence and progression of cancerous tumors. As a number of such mutations was identified, the therapies were developed that target the proteins encoded by the mutated genes, as well as the therapies targeting the signaling pathways in which these mutated genes are involved. As these targeted therapies were implemented into clinical practice, it was discovered that mutations conferring the resistance to the targeted therapies develop and accumulate in the patients' cancerous tumors, over time rendering the therapy ineffective and making it necessary to change the course of treatment.
One example of a solid tumor cancer in which somatic tumor mutations are known to play an important role is lung cancer, which is a leading cause of cancer-related mortality in many countries, including the United States. Approximately 75% of lung cancer cases belong to non-small cell lung cancer (NSCLC), which has an overall 5-year survival rate of approximately 12%. Standard surgical treatment, as well as chemotherapy and radiotherapies are available in the field of NSCLC. However, the majority of the NSCLC cases are initially diagnosed at the inoperable late stage, and relapse is common following surgery, chemotherapy, radiotherapy and other treatments. Accordingly, treatment and diagnosis of NSCLC is a challenging medical problem. One attempt at addressing the problem was the development of the targeted drug therapies that interfere with the signaling of epidermal growth factor receptor (EGFR). EGFR, which is a member of the growth factor receptor family of tyrosine kinases, is involved in signaling pathways related to cell division and is implicated in NSCLC development and progression.
Small molecule drugs erlotinib and gefitinib, which inhibit tyrosine kinase activity of EGFR, were evaluated and approved for treatment of late stage NSCLC. It was discovered, however, that these drugs were net effective in the majority of NSCLC patients, but are most effective in a subset of patients whose tumors contain somatic EGFR mutations that lead to an increase in the tyrosine kinase activity of EGFR. This type of mutations is often termed “activating.” Somatic EGFR mutations that lead to resistance to tyrosine kinase inhibitor therapy in NSCLC patients were also discovered. This type of mutations is often termed “resistance.” Resistance mutations in EGFR tend to arise in NSCLC patients during the course of tyrosine kinase inhibitor treatment. In the cases of NSCLC that cannot be effectively treated by tyrosine kinase inhibitor therapy, such as erlotinib and gefitinib, chemotherapy remains the most effective treatment to prolong survival. To improve the chances of selecting an effective treatment for NSCLC patients, it is therefore important to determine whether the patients' NSCLC tumors contain somatic EGFR mutations that confer sensitivity or resistance to tyrosine kinase inhibitor therapy.
The above example of the role of EGFR somatic mutations in the development of NSCLC illustrates how detecting the presence or emergence of certain mutations in the cancerous tumors is generally important for choosing an effective cancer treatment. For example, detecting the presence or emergence of somatic mutations leading to targeted drug therapy resistance in cancer patients is essential for monitoring the therapy and assessing disease progression. Therefore, it is generally beneficial to develop convenient and reliable methods of testing for somatic mutations in the tumors of the cancer patients in order to improve cancer assessment, including, but not limited to, diagnostics, monitoring, and treatment selection in such patients.
One way of detecting such mutations is testing tumor samples obtained through biopsy or surgery for the presence of mutant sequences associated with cancer development. However, tumor tissue samples may not be immediately available for testing. To avoid delay in detection of the cancer-associated mutations and selection of appropriate treatment as well as to spare the patients from invasive procedures, it is beneficial to develop more expedient and less invasive methods for detecting mutations in the tumors of the cancer patients.
It is known that tumor cells circulate in the blood of patients with solid tumor cancers, thus making it possible to detect somatic tumor mutations in the blood samples of cancer patients, including detection of EGFR mutations in NSCLC patients. However, it is difficult to reliably adapt such detection for meaningful clinical and diagnostic use due to the small amount of circulating mutated sequences, background of non-mutated sequences and high levels of genomic DNA (gDNA) circulating in the blood, the gDNA originating from broken white blood cells (WBC). Detection of mutated nucleic acid sequences originating from tumor cells in blood samples, such as detection of EGFR mutations in NSCLC patients, suffers from inaccuracies, such as relatively high false negative detection rates, and often requires cumbersome analytical techniques that may involve, for example, isolation of blood-circulating tumor cells prior to detection, or enrichment of the content of mutated DNA sequences in the sample prior to detection. Quantitative detection can be even more difficult, due to high background DNA levels, among other things. It is therefore important to develop improved methods of detection of mutated tumor nucleic acid sequences in the blood of cancer patients, such as detection of mutated EGFR nucleic acid sequences in the blood of NSCLC patients, to make such detection methods useful for assessment of cancer in clinical and diagnostic practice.